The overall objective of my research is to characterize the structure and function of components of the cell machinery responsible for DNA replication; the specific component to be examined in this project is the replication-specific enzyme, DNA polymerase III (pol III) of B. subtilis. My approach includes: (1) the examination of enzyme structure through the use of novel, pol III-specific molecular probes and classical techniques of protein chemistry, and (2) the examination of the structure and function of pol III in replicative synthesis, using mutant enzymes and a bacteriophage system (SPP-1) which requires pol III to replicate its DNA. The study of pol III structure, which will exploit homogeneous preparations of enzyme, will examine specifically: (1) the physical characteristics of the enzyme, its peptide map, content, antigenic nature, and structural features which may be common to replication-specific polymerases from other prokaryotic and eukaryotic systems; (2) the site of substrate binding, and (3) the site of reaction with the pol III-specific arylhydrazinopyrimidine inhibitors in an approach using wild-type and drug-resistant enzymes, irreversible inhibitor analogs, and peptide mapping. The study of pol III function will exploit mutant enzymes and will examine specifically: (1) the role of the nuclease and polymerase activities of pol III in the editing and synthesis of DNA, (2) the nature of replication-specific forms of pol III and their interactions with other host-specific and virus-specific proteins required for replication of SPP-1 in vivo and in vitro.